Widefield fluorescence macroscopy with out-of-focus blur rejection

描述(申请人提供)：我们的目标是开发一种新型的成像“宏观”，在厚组织中执行超深(几毫米)的荧光成像，同时提供焦外背景排斥。我们提出的抑制离焦模糊的方法是基于我们实验室最近发明的一种名为动态散斑照明(DSI)显微镜的新技术。我们的目标是建造一台便携式DSI宏观显微镜，并建立其在体内分子成像应用的潜力，例如癌症研究和诊断，以及小动物成像和手术。DSI显微镜是对标准宽视场荧光显微镜的简单改进。使用随机激光散斑图案而不是使用灯来执行样本照明。DSI显微镜的主要优点是在不使用复杂的扫描机制的情况下，在厚组织中提供深度分辨和焦外模糊减少。我们已经发展了DSI显微镜的完整理论，并已经证明了亚微米分辨率的成像与GFP标记的神经元的共焦样背景排斥反应，在小鼠的大脑中低至约100？m。我们建议开发一种新的仪器，它可以进行更深层次的成像，但分辨率较低。我们的目标深度是几毫米，也许是一厘米。为了实现这一目标，我们建议对我们的DSI显微镜进行重大重新设计，以纳入长工作距离远程中心物镜、近红外激光照明和更灵敏的CCD摄像机。我们还计划将DSI与结构照明对比度和多光谱成像相结合(对于后者，我们将与剑桥研究与仪器公司合作)。与传统的商用显微镜相比，我们的DSI显微镜的明显优势在于，DSI提供了焦外背景抑制，从而能够更好地定位和可视化厚组织中的标记结构。我们的DSI原型的初步测试将在放射学实验室提供的组织模型上进行。具有焦外模糊抑制的宽场荧光宏观成像。公共卫生：我们建议开发一种简单且廉价的设备，用于小动物癌症模型的成像，该设备执行具有焦外模糊排斥的超深荧光成像。我们的目标是建立一种新的癌症研究工具，最终可以在临床上实施。

DESCRIPTION (provided by applicant): Our aim is to develop of a new type of imaging "macroscope" that performs ultra-deep (several millimeters) fluorescence imaging in thick tissue while providing out-of-focus background rejection. Our proposed method for out-of-focus blur rejection is based on a novel technique called Dynamic Speckle Illumination (DSI) microscopy that was recently invented in our lab. Our goal is to build a portable DSI macroscope and establish its potential for in-vivo molecular-imaging applications such as cancer research and diagnosis, as well as small animal imaging and surgery. DSI microscopy consists of a simple modification to a standard widefield fluorescence microscope. Sample illumination is performed with random laser speckle patterns rather than with a lamp. The main advantage of DSI microscopy is that it provides depth discrimination and out-of-focus blur reduction in thick tissues without the use of a complicated scanning mechanism. We have developed the full theory of DSI microscopy and already demonstrated sub-micron-resolution imaging with confocal-like background rejection of GFP-labeled neurons, down to about 100¿m in mice brain. We propose to develop a new instrument that performs much deeper imaging but with lower resolution. Our targeted depth is several millimeters, down to perhaps a centimeter. To attain this goal we propose to significantly re-design of our DSI microscope to incorporate a long working-distance telecentric objective, near-infrared laser illumination, and a more sensitive CCD camera. We also plan to combine DSI with structured illumination contrast and multipsectral imaging (for the latter, we will work with Cambridge Research & Instrumentation). The defining advantage of our DSI macroscope compared to conventional commercially available macroscopes will be that DSI provides out-of-focus background rejection, enabling significantly better localization and visualization of labeled structures within thick tissue. Initial testing of our DSI prototype will be performed on tissue phantoms provided by a radiology lab. Widefield fluorescence macroscopy with out-of-focus blur rejection. Public health: We propose to develop a simple and inexpensive device that performs ultra-deep fluorescence imaging with out-of-focus blur rejection, for small animal cancer-model imaging. Our goal is to build a new tool for cancer research that can eventually be implemented in the clinic.